Methods of differentiating preadipocytes and uses thereof

ABSTRACT

Described herein are methods of transdifferentiating preadipocytes, populations of transdifferentiated preadipocytes, and methods of using the transdifferentiated preadipocytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62/218,719, filed on Sep. 15, 2015, entitled “DIRECT CONVERSION OF HUMAN PREADIPOCYTES INTO HEMATOPOIETIC, NEURONAL AND PANCREATIC ALPHACELLS BY OCT4 AND KLF4 OVEREXPRESSION,” the contents of which is incorporated by reference herein in its entirety.

This application also claims the benefit of and priority to U.S. Provisional Patent Application No. 62/294,646, filed on Feb. 12, 2016, entitled “METHODS OF DIFFERENTIATING PREADIPOCYTES AND USES THEREOF,” the contents of which is incorporated by reference herein in its entirety

SEQUENCE LISTING

This application contains a sequence listing filed in electronic form as an ASCII.txt file entitled 2222251010_ST25.bd, created on Sep. 15, 2015. The content of the sequence listing is incorporated herein in its entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.

FIG. 1 shows one embodiment of a method of transdifferentiating preadipocytes.

FIGS. 2A-2D show micrograph images of colony formation during transdifferentiation and reprogramming of human preadipocytes by lentiviral overexpression.

FIG. 3 shows a graph demonstrating principal component analysis on gene expression data of microarray displays distinct cell populations (preadipocyte, OK; single clone, OK_pool; mixed colonies, AdiPS; iPS cell) and clustering of replicates (n=2).

FIG. 4 shows a graph demonstrating relative signal intensity from microarray displays no significant changes in pluripotent markers that highly expressed in the fully reprogrammed AdiPS cells.

FIG. 5 shows a graph demonstrating relative signal intensity from microarray displays down regulation of fibroblast markers while the expression of EMT activators (Slug, N-Cad) maintains highly.

FIG. 6 shows a diagram of BioGPS gene annotation in transdifferentiated preadipocytes expressing ectopic Oct4 and Klf4. The number of lineage and tissue specific genes is summarized.

FIG. 7 shows a graph demonstrating relative signal intensity from microarray displays upregulation of blood cell specific gene expression from two factor induced OK cell (Single clone) and OK_pool (mixed colonies).

FIG. 8 shows a graph demonstrating brain specific gene expression in transdifferentiated preadipocytes.

FIG. 9 shows a graph demonstrating pancreatic progenitor and islet specific gene expression in transdifferentiated preadipocytes.

FIGS. 10A-10O show graphs demonstrating expression of genes GCG (FIG. 10A), IRX (FIG. 10B), PCSK1 (FIG. 10C), PDK4 (FIG. 10D), RGS4 (FIG. 10E), CNTN1 (FIG. 10F), LPPR4 (FIG. 10G), LOXL2 (FIG. 10H), KCTD12 (FIG. 10I), KL (FIG. 10J), ISL1 (FIG. 10K), HDAC9 (FIG. 10L), KCNJ2 (FIG. 10M), CD36 (FIG. 10N), FABP4 (FIG. 10O) from transdifferentiated preadipocytes.

FIGS. 11A-11D show images demonstrating results of immunocytochemistry of transdifferentiated pancreatic alpha cells expressing ectopic Oct4 and Klf4. The Oct4 and Klf4 expressing cells were seeded on matrigel plates and subjected to immunocytochemistry using glucagon (FIGS. 11A and 11B) and NeuroD (FIGS. 11C and 11D) specific antibodies and DAPI nuclear counterstaining.

FIG. 12 shows one embodiment of a method to transdifferentiation and reprogramming of human preadipoctyes to pancreatic alpha cells by Oct4 lentiviral overexpression.

FIGS. 13A-13D show micrograph images of colony formation during transdifferentiation and reprogramming of human preadipocytes by lentiviral overexpression.

FIGS. 14A-14N show graphs demonstrating gene expression as measured by real-time PCR of GCG (FIG. 14A), KL (FIG. 14B), LPPR4 (FIG. 14C), PDK4 (FIG. 14D), CNTN1 (FIG. 14E), KCTD12 (FIG. 14F), LOXL2 (FIG. 14G), PCSK1 (FIG. 14H), IRX2 (FIG. 14I), ISL1 (FIG. 14J), HDAC9 (FIG. 14K), KCNJ2 (FIG. 14L), CD36 (FIG. 14M), and FABP4 (FIG. 14N) in Oct4 induced pancreatic alpha cells (Oct4#1 and Oct4#5; single clones, Oct4, mixed colonies).

FIGS. 15A-15D show micrograph images demonstrating immunocytochemistry of transdifferentiated pancreatic alpha cells expressing ectopic Oct4. The Oct4 induced pancreatic alpha cells were seeded on matrigel plates and subjected to immunocytochemistry using glucagon (FIGS. 15A-15B) and NeuroD (FIGS. 15C-15D) specific antibodies and DAPI nuclear counterstaining.

FIGS. 16A-16C show micrograph images of preadipocytes that have been transdifferentiated down adipogenic (FIG. 16A), chondrogenic (FIG. 16B), and osteogenic (FIG. 16C) lineages.

FIGS. 17A-17D show fluorescent micrograph images of 2 factor (OK) and 4 factor (OKSM) transfected cells using highly proliferative, low passage preadipocytes (<3 passages).

FIG. 18 shows a graph demonstrating the percent of GFP positive cells in the cell populations shown in FIGS. 17A-17D, which indicates lentiviral transduction rates.

FIG. 19 shows a graph demonstrating expression of hematopoietic transcription factors (CD45+ cells) in preadipocytes, OK cells, and OK_pool.

FIG. 20 shows a graph demonstrating expression of hematopoietic transcription factors (Oct4+ cells) in preadipocytes, OK cells, and OK_pool.

FIG. 21 shows a graph demonstrating expression of hematopoietic cytokines in preadipocytes, OK cells, and OK_pool.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of molecular biology, microbiology, nanotechnology, organic chemistry, biochemistry, botany and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

Definitions

As used herein, “about,” “approximately,” and the like, when used in connection with a numerical variable, generally refers to the value of the variable and to all values of the variable that are within the experimental error (e.g., within the 95% confidence interval for the mean) or within ±10% of the indicated value, whichever is greater.

As used herein, “nucleic acid” and “polynucleotide” generally refer to a string of at least two base-sugar-phosphate combinations and refers to, among others, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. “Polynucleotide” and “nucleic acids” also encompasses such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. For instance, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. “Polynucleotide” and “nucleic acids” also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids. Natural nucleic acids have a phosphate backbone, artificial nucleic acids may contain other types of backbones, but contain the same bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “nucleic acids” or “polynucleotide” as that term is intended herein.

As used herein, “deoxyribonucleic acid (DNA)” and “ribonucleic acid (RNA)” can refer to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. RNA may be in the form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, RNAi (RNA interference construct), siRNA (short interfering RNA), or ribozymes.

As used herein, “nucleic acid sequence” and “oligonucleotide” also encompasses a nucleic acid and polynucleotide as defined above.

As used herein, “DNA molecule” includes nucleic acids/polynucleotides that are made of DNA. Similarly, “RNA molecule” includes nucleic acids/polynucleotides that are made of RNA.

As used herein, “gene” refers to a hereditary unit corresponding to a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a characteristic(s) or trait(s) in an organism. The term “gene” encompasses specific nucleotide sequences of a genome that are transcribed into an RNA product and are not translated into a protein as well as those genomic sequences that are transcribed into an RNA product yet are translated into a protein.

As used herein, the terms “exogenous DNA,” “exogenous nucleic acid sequence,” “exogenous polynucleotide,” “exogenous gene (including specifically named genes)” are used interchangeably with the terms “ectopic DNA,” “ectopic nucleic acid sequence,” “ectopic polynucleotide,” “ectopic gene (including specifically named genes)” and refer to a nucleic acid sequence that was introduced into a cell, organism, or organelle via transfection. Exogenous nucleic acids originate from an external source, for instance, the exogenous nucleic acid may be from another cell or organism and/or it may be synthetic and/or recombinant. While an exogenous nucleic acid sometimes originates from a different organism or species, it may also originate from the same species (e.g., an extra copy or recombinant form of a nucleic acid that is introduced into a cell or organism in addition to or as a replacement for the naturally occurring nucleic acid). Typically, the introduced exogenous sequence is a recombinant sequence.

As used herein, the term “recombinant” generally refers to a non-naturally occurring nucleic acid, nucleic acid construct, or polypeptide. Such non-naturally occurring nucleic acids may include natural nucleic acids that have been modified, for example that have deletions, substitutions, inversions, insertions, etc., and/or combinations of nucleic acid sequences of different origin that are joined using molecular biology technologies (e.g., a nucleic acid sequences encoding a “fusion protein” (e.g., a protein or polypeptide formed from the combination of two different proteins or protein fragments), the combination of a nucleic acid encoding a polypeptide to a promoter sequence, where the coding sequence and promoter sequence are from different sources or otherwise do not typically occur together naturally (e.g, a nucleic acid and a constitutive promoter), etc.). Recombinant also refers to the polypeptide encoded by the recombinant nucleic acid. Non-naturally occurring nucleic acids or polypeptides include nucleic acids and polypeptides modified by man.

As used herein, “polypeptides” or “proteins” are as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).

As used herein, “plasmid” as used herein refers to a non-chromosomal double-stranded DNA sequence including an intact “replicon” such that the plasmid is replicated in a host cell.

As used herein, the term “vector” or is used in reference to a vehicle used to introduce an exogenous nucleic acid sequence into a cell. A vector may include a DNA molecule, linear or circular (e.g. plasmids), which includes a segment encoding a polypeptide of interest operatively linked to additional segments that provide for its transcription and translation upon introduction into a host cell or host cell organelles. Such additional segments may include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from yeast or bacterial genomic or plasmid DNA, or viral DNA, or may contain elements of both.

As used herein, “promoter” includes all sequences capable of driving transcription of a gene. In particular, the term “promoter” as used herein can refer to a DNA sequence generally described as the 5′ regulator region of a gene, located proximal to the start codon. The transcription of an adjacent gene sequence is initiated at the promoter region. The term “promoter” also includes fragments of a promoter that are functional in initiating transcription of the gene. The term “promoter” can encompass constitutive promoters and inducible promoters.

As used herein, “constitutive promoter” is a promoter that allows for continual or ubiquitous transcription of its associated gene or polynucleotide. Constitutive promoters are generally are unregulated by cell or tissue type, time, or environment.

As used herein, “inducible promoter” is a promoter that allows transcription of its associated gene or polynucleotide in response to a substance or compound (e.g. an antibiotic, or metal), an environmental condition (e.g. temperature), developmental stage, or tissue type.

As used herein, “wild-type” is the average form of an organism, variety, strain, gene, protein, or characteristic as it occurs in a given population in nature, as distinguished from mutant forms that may result from selective breeding, recombinant engineering, and/or transformation with a transgene.

As used herein, “operatively linked” indicates that the regulatory sequences useful for expression of the coding sequences of a nucleic acid are placed in the nucleic acid molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements), and/or selectable markers in an expression vector.

As used herein, “expression” as used herein describes the process undergone by a structural gene to produce an RNA molecule and/or polypeptide. It can refer to the combination of transcription and translation. Expression can refer to the “expression” of a nucleic acid to produce a RNA molecule and can also refers to “expression” of a polypeptide, indicating that the polypeptide is being produced via expression of the corresponding nucleic acid.

As used herein, “control” is an alternative subject or sample used in an experiment for comparison purposes and included to minimize or distinguish the effect of variables other than an independent variable. A “control” can be a positive control, a negative control, or an assay or reaction control (an internal control to an assay or reaction included to confirm that the assay was functional). In some instances, the positive or negative control can also be the assay or reaction control.

As used interchangeably herein, “subject,” “individual,” or “patient,” refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. The term “pet” includes a dog, cat, guinea pig, mouse, rat, rabbit, ferret, and the like. The term farm animal includes a horse, sheep, goat, chicken, pig, cow, donkey, llama, alpaca, turkey, and the like.

As used herein, “passage,” “passaging” and the like, is a term of art that, in the context of cell culture, can refer to the process of subculturing a population of cells and includes physically removing a subset of cells from a cell population and expanding the subset separately from the original population in a fresh culture environment. As used herein “passaging” does not include simple media changes where no subset of the original population is isolated and propagated.

As used herein, “expansion” or “expanded” in the context of cells, refers to an increase in the number of a characteristic cell type, or cell types, from an initial population of cells, which may or may not be identical. The initial cells used for expansion need not be the same as the cells generated from expansion. For instance, the expanded cells may be produced by ex vivo or in vitro growth and differentiation of the initial population of cells. Expansion can also refer to allowing a cell population to undergo one or more cell division without passaging the cells.

As used herein, the abbreviation “Oct4” can refer to octamer-binding transcription factor 4 gene, gene product, and/or protein. Oct4 can be involved in the self-renewal of undifferentiated embryonic stem cells. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the Oct4 in humans and animals. For example, the Oct4 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession numbers NM_203289, NM_001173531, NM_001285986, NM_001285987.1, NM_002701, NM_001252452, and NM_013633. Other mRNA sequences that can be translated to form an Oct4 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, Oct4 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession numbers NP_001272916.1, NP_002692.2, NP_001272915.1, and NP_038661.2. Other Oct4 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “Klf4”, can refer to krupppel-like factor 4 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the Klf4 in humans and animals. For example, the Klf4 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession numbers NM_001314052 and NM_010637. Other mRNA sequences that can be translated to form a Klf4 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, Klf4 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession numbers NP_004226.3, NP_001300981.1, and NP_034767.2. Other Klf4 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein the abbreviation “Sox2” can refer to SRY(sex determining region Y)-box 2 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the Sox2 in humans and animals. For example, the Sox2 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession numbers NM_003106 and NM_011443. Other mRNA sequences that can be translated to form a Sox2 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, Sox2 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession numbers NP_003097.1 and NP_035573.3. Other Sox2 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein the abbreviation “cMyc” can refer to the Myc molecule gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the cMyc in humans and animals. For example, the cMyc mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession numbers NM_002467 and NM_001177352, NM_001177353, NM_001177354, and NM_010849. Other mRNA sequences that can be translated to form a cMyc protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, cMyc protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession numbers NP_002458.2, NP_001170823.1, NP_001170824.1, and NP_001170825.1. Other cMyc polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CNTN1” can refer to contactin 1 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the CNTN1 in humans and animals. For example, the CNTN1 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession numbers NM_001256063 and NM_001256064, NM_001843, NM_175038, NM_001159647, NM_001159648, and NM_007727. Other mRNA sequences that can be translated to form a CNTN1 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, CNTN1 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession numbers NP_001242992.1, NP_001242993.1, NP_001834.2, NP_778203.1, NP_001153119.1, NP_001153120.1, NP_031753.1. Other CNTN1 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “PCSK1” can refer to proprotein convertase 1 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the PCSK1 in humans and animals. For example, the PCSK1 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession numbers NM_001177876.1, NM_000439, NM_001177875, and NM_013628. Other mRNA sequences that can be translated to form a PCSK1 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, PCSK1 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession numbers NP_000430.3, NP_001171346.1, and NP_038656.1. Other PCSK1 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “PDK4” can refer to pyruvate dehydrogenase kinase isozyme 4 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the PDK4 in humans and animals. For example, the PDK4 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_002612. Other mRNA sequences that can be translated to form a PDK4 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, PDK4 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_002603.1. Other PDK4 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “RGS4” can refer to regulator of G protein signaling gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the RGS4 in humans and animals. For example, the RGS4 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001102445, NM_001113380, NM_001113381, NM_005613, and NM_009062. Other mRNA sequences that can be translated to form a RGS4 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, RGS4 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001095915.1, NP_001106851.1, NP_001106582.1, NP_005604.1, and NP_033088.2. Other RGS4 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “IRX2” can refer to Iroquois-class homeodomain gene and/or protein gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the IRX2 in humans and animals. For example, the IRX2 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001134222.1, NM_033267.4, and NM_010574. Other mRNA sequences that can be translated to form an IRX2 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, IRX2 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001127694.1, NP_150366.1, and NP_034704.1 Other IRX2 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “LPPR4” can refer to phospholipid phosphatase related 4 (also known as lipid phosphate phosphatase-related protein type 4) gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the LPPR4 in humans and animals. For example, the LPPR4 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001166252.1 and NM_014839.4. Other mRNA sequences that can be translated to form a LPPR4 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, LPPR4 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001159724.1 and NP_055654.2. Other LPPR4 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “LOXL2” can refer to lysyl oxidase like 2 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the LOXL2 in humans and animals. For example, the LOXL2 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_002318.2. Other mRNA sequences that can be translated to form a LOXL2 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, LOXL2 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_002309.1. Other LOXL2 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “LOXL4” can refer to lysyl oxidase like 4 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the LOXL4 in humans and animals. For example, the LOXL4 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_032211.6. Other mRNA sequences that can be translated to form a LOXL4 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, LOXL4 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_115587.6. Other LOXL4 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “KCTD12” can refer to the potassium channel teteramerization domain containing 12 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the KCTD12 in humans and animals. For example, the KCTD12 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_138444.3. Other mRNA sequences that can be translated to form a KCTD12 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, KCTD12 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_612453.1. Other KCTD12 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “KL” can refer to the Klotho gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the KL in humans and animals. For example, the KL mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_004795.3 and NM_153683.2. Other mRNA sequences that can be translated to form a KL protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, KL protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_004786.2. Other KL polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “RBPJ” can refer to recombination signal binding protein for immunoglobulin kappa J region gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the KL in humans and animals. For example, the KL mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_005349.3, NM_015874.4, NM_203283.2, and NM_203284.2. Other mRNA sequences that can be translated to form a KL protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, KL protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_005340.2, NP_056958.3, NP_976028.1, and NP_976029.1. Other KL polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “Sox9” can refer to SRY-Box 9 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the Sox9 in humans and animals. For example, the Sox9 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_000346.3. Other mRNA sequences that can be translated to form a Sox9 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, Sox9 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_000337.1. Other Sox9 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “NeuroD” can refer to neuronal differentiation 1 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the NeuroD in humans and animals. For example, the NeuroD mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_002500.4. Other mRNA sequences that can be translated to form a NeuroD protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, NeuroD protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_002491.2. Other NeuroD polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “ISL1” can refer to ISL LIM Homeobox 1 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the ISL1 in humans and animals. For example, the ISL1 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_002202.2. Other mRNA sequences that can be translated to form an ISL1 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, ISL1 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_002193.2. Other ISL1 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “RGS2” can refer to regulator of G-Protein Signaling 2 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the RGS2 in humans and animals. For example, the RGS2 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_002923.3. Other mRNA sequences that can be translated to form a RGS2 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, RGS2 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_002914.1. Other RGS2 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “IL8” can refer to interleukin 8 (also known as C-X-C motif ligand 8) gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the IL8 in humans and animals. For example, the IL8 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_000584.3. Other mRNA sequences that can be translated to form an IL8 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, IL8 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_000575.1. Other IL8 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “AKAP7” can refer to A-kinase anchoring protein 7 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the AKAP7 in humans and animals. For example, the AKAP7 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_004842.3, NM_016377.3, and NM_138633.2. Other mRNA sequences that can be translated to form an AKAP7 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, AKAP7 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_004833.1, NP_057461.2, and NP_619539.1. Other AKAP7 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CPE” can refer to carboxypeptidase E gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the CPE in humans and animals. For example, the CPE mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001873.3. Other mRNA sequences that can be translated to form a CPE protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, CPE protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001864.1. Other CPE polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “OAS1” can refer to 2′-5′-Oligoadenylate Synthetase 1 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the OAS1 in humans and animals. For example, the OAS1 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001032409.2, NM_001320151.1, NM_002534.3, and NM_016816.3. Other mRNA sequences that can be translated to form an OAS1 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, OAS1 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001027581.1, NP_001307080.1, NP_002525.2, and NP_058132.2. Other OAS1 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “PLTP” can refer to phospholipid transfer protein gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the PLTP in humans and animals. For example, the PLTP mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001242920.1, NM_001242921.1, NM_006227.3, and NM_182676.2. Other mRNA sequences that can be translated to form a PLTP protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, PLTP protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001229849.1, NP_001229850.1, NP_006218.1, and NP_872617.1. Other PLTP polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “PGS4” can refer to leucine rich repeat containing 49 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the PGS4 in humans and animals. For example, the PGS4 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001199017.2, NM_001199018.2, NM_001284357.1, and NM_017691.4. Other mRNA sequences that can be translated to form a PGS4 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, PGS4 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001185946.1, NP_001185947.1, NP_001271286.1, and NP_060161.2. Other PGS4 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “RNF128” can refer to ring finger protein 128 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the RNF128 in humans and animals. For example, the RNF128 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_024539.3 and NM_194463.1. Other mRNA sequences that can be translated to form a RNF128 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, RNF128 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_078815.3 and NP_919445.1. Other RNF128 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “BEX1” can refer to brain expressed X-linked 1 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the BEX1 in humans and animals. For example, the BEX1 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_018476.3. Other mRNA sequences that can be translated to form a BEX1 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, BEX1 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_060946.3. Other BEX1 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “HDAC9” can refer to histone deacetylase 9 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the HDAC9 in humans and animals. For example, the HDAC9 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001204144.2, NM_001204145.2, NM_001204146.2, NM_001204147.2, and NM_001204148.2. Other mRNA sequences that can be translated to form a HDAC9 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, HDAC9 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001191073.1, NP_001191074.1, NP_001191075.1, NP_001191076.1, NP_001191077.1, NP_001308797.1, NP_001308798.1, NP_001308799.1, NP_001308800.1, NP_001308801.1, NP_001308802.1, NP_001308803.1, NP_001308804.1, NP_001308805.1, NP_001308806.1, NP_001308807.1, NP_001308808.1, NP_001308813.1, NP_001308814.1, NP_001308815.1, NP_001308816.1, NP_001308817.1, NP_001308818.1, NP_001308819.1, NP_001308820.1, NP_001308822.1, NP_001308823.1, NP_001308824.1, NP_001308825.1, NP_001308826.1, NP_001308827.1, NP_001308828.1, NP_001308829.1, NP_001308830.1, NP_001308831.1, NP_055522.1, NP_478056.1, and NP_848510.1, NP_848512.1. Other HDAC9 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “KCNJ2” cam refer to potassium voltage-gated channel subfamily J member 2 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the KCNJ2 in humans and animals. For example, the KCNJ2 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_000891.2. Other mRNA sequences that can be translated to form a KCNJ2 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, KCNJ2 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_000882.1. Other KCNJ2 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CD36” can refer to the CD36 molecule gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the CD36 in humans and animals. For example, the CD36 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_000072.3, NM_001001547.2, NM_001001548.2, NM_001127443.1, NM_001127444.1, NM_001289908.1, NM_001289909.1, and NM_001289911.1. Other mRNA sequences that can be translated to form a CD36 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, CD36 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_000063.2, NP_001001547.1, NP_001001548.1, NP_001120915.1, NP_001120916.1, NP_001276837.1, NP_001276838.1, and NP_001276840.1. Other CD36 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “FABP4” can refer to the fatty acid binding protein 4 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the FABP4 in humans and animals. For example, the FABP4 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001442.2. Other mRNA sequences that can be translated to form a FABP4 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, FABP4 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001433.1. Other FABP4 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CD71” can refer to the transferrin receptor gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the CD71 in humans and animals. For example, the CD71 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001128148.2, NM_001313965.1, NM_001313966.1, and NM_003234.3. Other mRNA sequences that can be translated to form a CD71 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, CD71 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001121620.1, NP_001300894.1, NP_001300895.1, and NP_003225.2. Other CD71 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CHST2” can refer to the carbohydrate sulfotransferase 2 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the CHST2 in humans and animals. For example, the CHST2 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_004267.4. Other mRNA sequences that can be translated to form a CHST2 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, CHST2 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_004258.2. Other CHST2 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “AMPD3” can refer to the adenosine monophosphate deaminase 3 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the AMPD3 in humans and animals. For example, the AMPD3 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_000480.2 NM_001025389.1 NM_001025390.1 NM_001172430.1 NM_001172431.1. Other mRNA sequences that can be translated to form an AMPD3 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, AMPD3 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_000471.1, NP_001020560.1, NP_001020561.1, NP_001165901.1, and NP_001165902.1. Other AMPD3 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CD58” can refer to the CD58 molecule gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the CD58 in humans and animals. For example, the CD58 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001144822.1 and NM_001779.2. Other mRNA sequences that can be translated to form a CD58 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, CD58 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001138294.1 and NP_001770.1. Other CD58 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “MINPP1” can refer to the multiple inositol-polyphosphate phosphatase 1 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the MINPP1 in humans and animals. For example, the MINPP1 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001178117.1, NM_001178118.1, and NM_004897.4. Other mRNA sequences that can be translated to form a MINPP1 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, MINPP1 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001171588.1, NP_001171589.1, and NP_004888.2. Other MINPP1 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “IDH2” can refer to the isocitrate dehydrogenase (NADP(+))2 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the IDH2 in humans and animals. For example, the IDH2 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001289910.1, NM_001290114.1, and NM_002168.3. Other mRNA sequences that can be translated to form an IDH2 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, IDH2 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001276839.1, NP_001277043.1, and NP_002159.2. Other IDH2 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CD70” can refer to the CD70 Molecule (also referred to as tumor necrosis factor (ligand) superfamily, member 70) gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the CD70 in humans and animals. For example, the CD70 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001252.4. Other mRNA sequences that can be translated to form a CD70 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, CD70 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001243.1. Other CD70 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “HLA-DRA” can refer to the major histocompatibility complex, Class II, DR alpha gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the HLA-DRA in humans and animals. For example, the HLA-DRA mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_019111.4. Other mRNA sequences that can be translated to form a HLA-DRA protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, HLA-DRA protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_061984.2. Other HLA-DRA polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “NX1” can refer to the neurexin 1 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the NX1 in humans and animals. For example, the NX1 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001135659.2, NM_001320156.2, NM_001320157.3, NM_004801.5, and NM_138735.4. Other mRNA sequences that can be translated to form a NX1 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, NX1 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001129131.1, NP_001307085.1, NP_001307086.1, NP_004792.1, and NP_620072.1. Other NX1 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CD83” can refer to the CD83 molecule gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the CD83 in humans and animals. For example, the CD83 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001040280.1, NM_001251901.1, and NM_004233.3. Other mRNA sequences that can be translated to form a CD83 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, CD83 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001035370.1, NP_001238830.1, and NP_004224.1. Other CD83 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CD74” can refer to the CD74 molecule gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the CD74 in humans and animals. For example, the CD74 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001025158.2, NM_001025159.2, and NM_004355.3. Other mRNA sequences that can be translated to form a CD74 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, CD74 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001020329.1, NP_001020330.1, and NP_004346.1. Other CD74 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “IFI44L” can refer to the interferon induced protein 44 like gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the IFI44L in humans and animals. For example, the IFI44L mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_006820.3. Other mRNA sequences that can be translated to form an IFI44L protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, IFI44L protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_006811.2. Other IFI44L polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “BST2” can refer to the bone marrow stromal cell antigen 2 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the BST2 in humans and animals. For example, the BST2 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_004335.3. Other mRNA sequences that can be translated to form a BST2 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, BST2 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_004326.1. Other BST2 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “ISG15” can refer to the ISG15 ubiquitin-like modifier gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the ISG15 in humans and animals. For example, the ISG15 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_005101.3. Other mRNA sequences that can be translated to form an ISG15 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, ISG15 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_005092.1. Other ISG15 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CLIC6” can refer to the chloride intracellular channel 6 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the CLIC6 in humans and animals. For example, the CLIC6 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001317009.1 and NM_053277.2. Other mRNA sequences that can be translated to form a CLIC6 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, CLIC6 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001303938.1 and NP_444507.1. Other CLIC6 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “PBX3” can refer to the pre-B-Cell Leukemia Homeobox 3 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the PBX3 in humans and animals. For example, the PBX3 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001134778.1 and NM_006195.5. Other mRNA sequences that can be translated to form a PBX3 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, PBX3 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001128250.1 and NP_006186.1. Other PBX3 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “TOX2” can refer to TOX high mobility group box family member 2 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the TOX2 in humans and animals. For example, the TOX2 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001098796.1, NM_001098797.1, NM_001098798.1, and NM_032883.2. Other mRNA sequences that can be translated to form a TOX2 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, TOX2 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001092266.1, NP_001092267.1, NP_001092268.1, and NP_116272.1. Other TOX2 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “HCP5” can refer to HLA Complex P5 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the HCP5 in humans and animals. For example, the HCP5 RNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NR_040662.1.

As used herein, the abbreviation “RAC2” can refer to RAC2 high mobility group box family member 2 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the RAC2 in humans and animals. For example, the RAC2 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_002872.4. Other mRNA sequences that can be translated to form a RAC2 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, RAC2 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_002863.1. Other RAC2 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “RGS1” can refer to Regulator of G-Protein Signaling 1 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the RGS1 in humans and animals. For example, the RGS1 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_002922.3. Other mRNA sequences that can be translated to form a RGS1 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, RGS1 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_002913.3. Other RGS1 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “STAT4” can refer to Regulator of G-Protein Signaling 1 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the STAT4 in humans and animals. For example, the STAT4 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001243835.1 and NM_003151.3. Other mRNA sequences that can be translated to form a STAT4 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, STAT4 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001230764.1 and NP_003142.1. Other STAT4 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “HLA-DMB” can refer to the Major Histocompatibility Complex, Class II, DM Beta gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the HLA-DMB in humans and animals. For example, the HLA-DMB mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_002118.4. Other mRNA sequences that can be translated to form a HLA-DMB protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, HLA-DMB protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_002109.2. Other HLA-DMB polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “NT5E” can refer to the 5′-Nucleotidase Ecto gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the NT5E in humans and animals. For example, the NT5E mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001204813.1 and NM_002526.3. Other mRNA sequences that can be translated to form a NT5E protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, NT5E protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001191742.1 and NP_002517.1. Other NT5E polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “BTG1” can refer to the B-Cell Translocation Gene 1 gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the BTG1 in humans and animals. For example, the BTG1 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001731.2. Other mRNA sequences that can be translated to form a BTG1 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, BTG1 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001722.1. Other BTG1 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CD14” can refer to the CD14 molecule gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the CD14 in humans and animals. For example, the CD14 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_000591.3. Other mRNA sequences that can be translated to form a CD14 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, CD14 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_000582.1. Other CD14 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art

As used herein, the abbreviation “F13A1” can refer to the Coagulation Factor XIII A Chain gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the F13A1 in humans and animals. For example, the F13A1 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_000129.3. Other mRNA sequences that can be translated to form a F13A1 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, F13A1 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_000120.2. Other F13A1 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “FGL2” can refer to the fibrinogen like 2 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the FGL2 in humans and animals. For example, the FGL2 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_006682.2. Other mRNA sequences that can be translated to form a FGL2 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, FGL2 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_006673.1. Other FGL2 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “FOLR3” can refer to the Folate Receptor 3 (Gamma) gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the FOLR3 in humans and animals. For example, the FOLR3 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_000804.3. Other mRNA sequences that can be translated to form a FOLR3 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, FOLR3 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_000795.2. Other FOLR3 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “MX1” can refer to the MX Dynamin like GTPase 1 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the MX1 in humans and animals. For example, the MX1 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001144925.2. Other mRNA sequences that can be translated to form a MX1 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, MX1 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001138397.1. Other MX1 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “PSENEN” can refer to the Presenilin Enhancer Gamma Secretase Subunit gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the PSENEN in humans and animals. For example, the RPSENEN mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_172341.2. Other mRNA sequences that can be translated to form a RSENEN protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, PSENEN protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_758844.1. Other PSENEN polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “IFI6” can refer to the Interferon Alpha Inducible Protein 6 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the IFI6 in humans and animals. For example, the IFI6 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_002038.3. Other mRNA sequences that can be translated to form an IFI6 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, IFI6 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_002029.3. Other IFI6 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “HS3ST2” can refer to the Heparan Sulfate-Glucosamine 3-Sulfotransferase 2 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the HS3ST2 in humans and animals. For example, the HS3ST2 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_006043.1. Other mRNA sequences that can be translated to form a HS3ST2 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, HS3ST2 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s NP_006034.1. Other HS3ST2 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “TAGLN3” can refer to the Transgelin 3 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the TAGLN3 in humans and animals. For example, the TAGLN3 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_013259.2. Other mRNA sequences that can be translated to form a TAGLN3 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, TAGLN3 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession numbers NP_037391.2. Other TAGLN3 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “SV2B” can refer to the Synaptic Vesicle Glycoprotein 2B gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the SV2B in humans and animals. For example, the SV2B mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) and NM_014848.6. Other mRNA sequences that can be translated to form a SV2B protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, SV2B protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_055663.1. Other SV2B polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “NCALD” can refer to the Neurocalcin Delta gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the NCALD in humans and animals. For example, the NCALD mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001040624.1. Other mRNA sequences that can be translated to form a NCALD protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, NCALD protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001035714.1. Other NCALD polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “ENC1” can refer to the Ectodermal-Neural Cortex 1 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the ENC1 in humans and animals. For example, the ENC1 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession numbers NM_003633.3. Other mRNA sequences that can be translated to form an ENC1 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, ENC1 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_003624.1. Other ENC1 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “COBL” can refer to the Cordon-Bleu WH2 Repeat Protein gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the COBL in humans and animals. For example, the COBL mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001287436.1. Other mRNA sequences that can be translated to form a COBL protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, COBL protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001274365.1. Other COBL polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “EEF1A2” can refer to the Eukaryotic Translation Elongation Factor 1 Alpha 2 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the EEF1A2 in humans and animals. For example, the EEF1A2 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001958.3. Other mRNA sequences that can be translated to form an EEF1A2 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, EEF1A2 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001949.1. Other EEF1A2 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “KBTBD11” can refer to the Kelch Repeat and BTB Domain Containing 11 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the KBTBD11 in humans and animals. For example, the KBTBD11 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001958.3. Other mRNA sequences that can be translated to form a KBTBD11 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, KBTBD11 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_055682.1. Other KBTBD11 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “RGL1” can refer to the Ral Guanine Nucleotide Dissociation Stimulator Like 1 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the RGL1 in humans and animals. For example, the RGL1 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_015149.4. Other mRNA sequences that can be translated to form a RGL1 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, RGL1 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_055964.3. Other RGL1 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CA11” can refer to the Carbonic Anhydrase 11 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the CA11 in humans and animals. For example, the CA11 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001217.4. Other mRNA sequences that can be translated to form a CA11 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, CA11 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001208.2. Other CA11 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “NETO2” can refer to the Neuropilin and Tolloid Like 2 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the NETO2 in humans and animals. For example, the NETO2 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_018092.4. Other mRNA sequences that can be translated to form a NETO2 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, NETO2 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_060562.3. Other NETO2 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “DOK5” can refer to the Docking Protein 5 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the DOK5 in humans and animals. For example, the DOK5 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_018431.4. Other mRNA sequences that can be translated to form a DOK5 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, DOK5 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_060901.2. Other DOK5 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “TUBB2B” can refer to the Tubulin Beta 2B Class IIb gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the TUBB2B in humans and animals. For example, the TUBB2B mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_178012.4. Other mRNA sequences that can be translated to form a TUBB2B protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, TUBB2B protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_821080.1. Other TUBB2B polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “PCDH19” can refer to the Protocadherin 19, gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the PCDH19 in humans and animals. For example, the PCDH19 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001105243.1. Other mRNA sequences that can be translated to form a PCDH19 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, PCDH19 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001098713.1. Other PCDH19 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “PTPRO” can refer to the Protein Tyrosine Phosphatase, Receptor Type O gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the PTPRO in humans and animals. For example, the PTPRO mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_030667.2. Other mRNA sequences that can be translated to form a PTPRO protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, PTPRO protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_109592.1. Other PTPRO polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “SATB2” can refer to the SATB Homeobox 2 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the SATB2 in humans and animals. For example, the SATB2 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001172509.1. Other mRNA sequences that can be translated to form a SATB2 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, SATB2 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001165980.1. Other SATB2 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “FGF13” can refer to the Fibroblast Growth Factor 13 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the FGF13 in humans and animals. For example, the FGF13 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_004114.3. Other mRNA sequences that can be translated to form a FGF13 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, FGF13 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_004105.1. Other FGF13 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CXADR” can refer to the Coxsackie Virus And Adenovirus Receptor gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the CXADR in humans and animals. For example, the CXADR mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001338.4. Other mRNA sequences that can be translated to form a CXADR protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, CXADR protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001329.1. Other CXADR polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “MLLT11” can refer to the Myeloid/Lymphoid or Mixed-Lineage Leukemia; Translocated To, 11 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the MLLT11 in humans and animals. For example, the MLLT11 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_006818.3. Other mRNA sequences that can be translated to form a MLLT11 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, MLLT11 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_006809.1. Other MLLT11 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “TMEFF2” can refer to the Transmembrane Protein with EGF Like and Two Follistatin like Domains 2 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the TMEFF2 in humans and animals. For example, the TMEFF2 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_016192.3. Other mRNA sequences that can be translated to form a TMEFF2 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, TMEFF2 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_057276.2. Other TMEFF2 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “BRSK1” can refer to the BR Serine/Threonine Kinase 1 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the BRSK1 in humans and animals. For example, the BRSK1 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_032430.1. Other mRNA sequences that can be translated to form a BRSK1 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, BRSK1 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_115806.1. Other BRSK1 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “NRGN” can refer to the Neurogranin gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the NRGN in humans and animals. For example, the NRGN mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_006176.2. Other mRNA sequences that can be translated to form a NRGN protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, NRGN protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_006167.1. Other NRGN polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “TAC1” can refer to the Tachykinin Precursor 1 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the TAC1 in humans and animals. For example, the TAC1 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_003182.2. Other mRNA sequences that can be translated to form a TAC1 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, TAC1 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_003173.1. Other TAC1 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CDH10” can refer to the Cadherin 10 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the CDH10 in humans and animals. For example, the CDH10 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_006727.4. Other mRNA sequences that can be translated to form a CDH10 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, CDH10 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_006718.2. Other CDH10 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “TPD52L1” can refer to the Tumor Protein D52-Like 1 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the TPD52L1 in humans and animals. For example, the TPD52L1 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_003287.3. Other mRNA sequences that can be translated to form a TPD52L1 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, TPD52L1 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_003278.1. Other TPD52L1 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “HIST2H2BE” can refer to the Histone Cluster 2, H2be gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the HIST2H2BE in humans and animals. For example, the HIST2H2BE mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_003528.2. Other mRNA sequences that can be translated to form a HIST2H2BE protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, HIST2H2BE protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_003519.1. Other HIST2H2BE polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “SLC16A9” can refer to the Solute Carrier Family 16 Member 9 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the SLC16A9 in humans and animals. For example, the SLC16A9 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_194298.2. Other mRNA sequences that can be translated to form a SLC16A9 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, SLC16A9 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_919274.1. Other SLC16A9 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “MEGF10” can refer to the Multiple EGF Like Domains 10 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the MEGF10 in humans and animals. For example, the MEGF10 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_032446.2. Other mRNA sequences that can be translated to form a MEGF10 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, MEGF10 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_115822.1. Other MEGF10 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “FMNL2” can refer to the Formin like 2 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the FMNL2 in humans and animals. For example, the FMNL2 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_052905.3. Other mRNA sequences that can be translated to form a FMNL2 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, FMNL2 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_443137.2. Other FMNL2 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “KIAA1598” can refer to the Shootin 1 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the KIAA1598 in humans and animals. For example, the KIAA1598 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001127211.2. Other mRNA sequences that can be translated to form a KIAA1598 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, KIAA1598 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001120683.1. Other KIAA1598 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “DNER” can refer to the Delta/Notch Like EGF Repeat Containing gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the DNER in humans and animals. For example, the DNER mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_139072.3. Other mRNA sequences that can be translated to form a DNER protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, DNER protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_620711.3. Other DNER polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “KAL1” can refer to the Anosmin 1 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the KAL1 in humans and animals. For example, the KAL1 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_000216.3. Other mRNA sequences that can be translated to form a KAL1 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, KAL1 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_000207.2. Other KAL1 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CD45” can refer to the Protein Tyrosine Phosphatase, Receptor Type C gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the CD45 in humans and animals. For example, the CD45 mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_002838.4. Other mRNA sequences that can be translated to form a CD45 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, CD45 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_002829.3. Other CD45 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “AMY1C” can refer to the Amylase, Alpha 1C (Salivary) gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the AMY1C in humans and animals. For example, the AMY1C mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_001008219.1. Other mRNA sequences that can be translated to form an AMY1C protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, AMY1C protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_001008220.1. Other AMY1C polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “AMY1A” can refer to the Amylase, Alpha 1A (Salivary) gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the AMY1A in humans and animals. For example, the AMY1A mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_004038.3. Other mRNA sequences that can be translated to form an AMY1A protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, AMY1A protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_004029.2. Other AMY1A polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “GCG” can refer to the glucagon gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the GCG in humans and animals. For example, the GCG mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_002054.4. Other mRNA sequences that can be translated to form a GCG protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, GCG protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_002045.1. Other GCG polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CEBPD” can refer to the CCAAT/Enhancer Binding Protein Delta gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the CEBPD in humans and animals. For example, the CEBPD mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_005195.3. Other mRNA sequences that can be translated to form a CEBPD protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, CEBPD protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_005186.2. Other CEBPD polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CEBPD” can refer to the CCAAT/Enhancer Binding Protein Delta gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the CEBPD in humans and animals. For example, the CEBPD mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_005195.3. Other mRNA sequences that can be translated to form a CEBPD protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, CEBPD protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_005186.2. Other CEBPD polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “STEAP3” can refer to the Six-Transmembrane Epithelial Antigen of Prostate 3 gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the STEAP3 in humans and animals. For example, the STEAP3mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) NM_182915.2. Other mRNA sequences that can be translated to form a STEAP3 protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, STEAP3 protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) NP_878919.2. Other STEAP3 polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “AMT1B” can refer to the ammonium transport protein 1B gene, gene product, and/or protein. One of ordinary skill in the art will instantly appreciate RNA and protein variants, homologues, orthologues, and analogues of the AMT1B in bacteria, fungi, humans, and animals. For example, the AMT1B mRNA can have a nucleotide sequence as specified by, but not limited to, any of GenBank Accession number(s) XM_001692619.1. Other mRNA sequences that can be translated to form an AMT1B protein or variant thereof in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art. Similarly, AMT1B protein can have a polypeptide sequences as specified by, but not limited to, any of GenBank Accession number(s) XM_001692619.1. Other AMT1B polypeptides in human or other animal species can be identified and detected by one of ordinary skill in the art using techniques generally known to one of ordinary skill in the art.

SEQ ID NOs: for the various accession numbers specifically provided herein can be found in Table 1. Other variants of the sequences that correspond with the genes, gene products, and proteins provided herein will be appreciated by those of ordinary skill in the art and are within the scope of this disclosure.

Discussion

In most tissues, including the pancreas, a mature cell performing a specialized function represents a terminally differentiated cell that is restricted in potential. In other words, most mature cells possess the ability to carry out specific functions but lack the ability to replicate and expand the pool of cells. In contrast, progenitor cells typically lack very specific functionality but have the capacity and plasticity to proliferate and expand a pool of cells. In some instances it is desirable, particularly for the development of in vitro culture systems for cells to exhibit specific functionalities attributed to a terminally differentiated cell yet continue to proliferate to allow for culture in vitro.

It was once believed that differentiation was a one-way fate. However, it is now known that it is possible to reprogram differentiated cells to cell having more plasticity that can be directed down a different differentiation pathway. Pancreatic cells (including alpha and beta cells) are derived from a common progenitor. Many examples exist of fate conversion between endocrine cell types. Further exocrine cells, particularly acinar cells, have been used to convert the exocrine cells into an endocrine lineage, specifically pancreatic beta cells.

A major, yet poorly understood, feature of type 2 diabetes is the excessive hepatic glucose production and the corresponding insulin resistance leading to fasting hyperglycemia. Recent work has demonstrated an increased daily and unregulated plasma glucagon in concentration in type 2 diabetics. Glucagon secretion by pancreatic alpha-cells is an immediate response to glucopenia. Abnormal secretion of glucagon and other counterregulatory hormones is a hallmark of types 1 and 2 diabetes and a major limitation to the use of strong hypoglycemia agents. As such there exists a need for improved diabetes treatments, of which some may target the pancreatic alpha cells and dysregulation of glucagon secretion.

With that said, described herein are methods of transdifferentiating preadipocytes into, inter alia, pancreatic cells (e.g. pancreatic alpha cells), populations of transdifferentiated preadipocytes, and assays using the transdifferentiated preadipocytes to evaluate the effect of candidate compounds on a characteristic of the transdifferentiated preadipocyte.

Other compositions, compounds, methods, features, and advantages of the present disclosure will be or become apparent to one having ordinary skill in the art upon examination of the following drawings, detailed description, and examples. It is intended that all such additional compositions, compounds, methods, features, and advantages be included within this description, and be within the scope of the present disclosure.

Methods of Transdifferentiating Preadipocytes

Described herein are methods of transdifferentiating preadipocytes. Preadipocytes can be differentiated into hematopoietic, neuronal, and/or pancreatic cells. In some embodiments, the preadipocytes can be transdifferentiated into pancreatic alpha cells. In some embodiments, preadipocytes can be transdifferentiated by overexpressing Oct4 in the preadipocytes. In some embodiments, the preadipocytes can be transdifferentiated by overexpressing Oct4 with at least one other factor from the group of Klf4, Sox2, and cMyc. In some embodiments, the preadipocytes can be transdifferentiated by overexpressing Oct4 and Klf4. In some embodiments, the preadipocytes can be transdifferentiated by overexpressing Oct4, Klf4, and Sox2. In some embodiments, the preadipocytes can be transdifferentiated by overexpressing Oct4, Klf4, Sox2, and cMyc. In embodiments where Oct4 is overexpressed in the preadipocyte with at least one other factor from the group of Klf4, Sox2, and cMyc, the additional factor(s) can be overexpressed at the same (simultaneously) or at different times than the Oct4. In some embodiments, preadipocytes can be transdifferentiated without overexpressing Klf4, Sox2, and/or cMyc. Overexpression of Oct4,

In some embodiments, preadipocytes can be transdifferentiated by overexpressing Oct 4, Klf4, Sox2, and/or cMyc in the preadipocytes followed by culturing the cells until about 100% confluent, after which, the cells can be passaged and a subset of cells can be reseeded onto a feeder cell layer. The cells can then be expanded on the feeder layer until the desired differentiated cell type is achieved.

During the first culture period, cells can be cultured in a preadipocyte growth medium. In some embodiments the preadipocyte growth medium can contain Dulbecco's modified Eagle medium (DMEM) supplemented with FBS at about 10% and suitable antibiotics. In embodiments, after passaging, cells or subset thereof can be reseeded and expanded on the feeder layer in a suitable stem cell culture medium, such as, but not limited to, mTeSR1 media. In some embodiments, the first culture period can be anywhere from about 1-7 or more days. In other words, the cells can be cultured after transfection or transduction for about 1-7 days without passaging. In some embodiments, the cells can be expanded on the feeder layer for about 1-10 days.

Oct4, Klf4, Sox2, and/or cMyc can be ectopically (or exogenously) expressed or overexpressed in preadipocytes by any suitable method. Suitable methods include, but are not limited to viral-based and non-viral based transduction and transfection techniques. Viral based techniques can include those that involve adenoviral, lentiviral, and retroviral systems. Non-virus based techniques include liposomal and micelle delivery reagents. In some embodiments, the transgene can be incorporated into the genome utilizing CRISPR or other genome modification technique known by those of skill in the art. In some embodiments, expression or overexpression of Oct4, Klf4, Sox2, and/or cMyc can be transient or stable. In some embodiments, expression or overexpression of Oct4, Klf4, Sox2, and/or cMyc can be inducible. The preadipocytes can be transfected or transduced 1, 2, 3, 4, or more times with the same or different transgene(s).

Preadipocytes can be harvested from adipose tissue. In some embodiments, the preadipocytes can be obtained from the stromal vascular fraction of adipose tissue. In some embodiments the preadipocytes that are transfected or transduced are low passage preadipocytes. As used herein, “low passage” can refer to cells that have undergone 3 or less passages.

Transdifferentiated Cells

Also described herein are cells derived from transdifferentiated from preadipocytes according to the methods described herein. Insofar as these cells are transdifferentiated by at least exogenous expression of Oct4, these cells are not identical to preadipocytes or differentiated cells even if there are similarities in function or structure to a native or natural cell, such as an isolated preadipocyte or pancreatic cell, and thus not a product of nature.

In some embodiments, the transdifferentiated cells (also referred to herein as transdifferentiated preadipocytes) can have ectopic expression of Oct4. The transdifferentiated preadipocytes can also have ectopic expression of Klf4, Sox2, and/or cMyc. In some embodiments the transdifferentiated preadipocytes only have ectopic expression of Oct4. In further embodiments, the transdifferentiated preadipocytes only have ectopic expression of Oct4 and Klf4. Stated differently, in some embodiments, the transdifferentiated preadipocytes having ectopic expression of Oct4 do not have ectopic expression of Klf4, Sox2, and/or cMyc.

While transdifferentiated preadipocytes can be similar in function, genotype, phenotype, and/or structure to native pancreatic cells (e.g. pancreatic alpha or beta cells), neuronal cells, and/or hematopoietic cells, the populations of cells provided herein are not native or natural “isolated” pancreatic cells, neuronal cells, and/or hematopoietic cells insofar as they can have differing expression patterns (or profiles) of genes and/or proteins as described herein and/or have exogenous expression of one or more genes and/or proteins (e.g. Oct4) as described herein. These non-natural cells can be referred to herein as pancreatic cells, neuronal cells, and hematopoietic cells.

In some embodiments the transdifferentiated preadipocytes can express NeuroD protein. In this way, the transdifferentiated preadipocytes can be similar to pancreatic alpha cells. In some embodiments, the transdifferentiated preadipocytes have increased expression of a pancreatic alpha cell specific gene as compared to an unmodified preadipocyte. The pancreatic alpha cell specific gene that can control pancreatic hormone expression and processing can be any one of GCG, CNTN1, PCSK1, PDK4, RGS4, IRX2, LPPR4, LOXL2, KCTD12, KL, and any combination thereof. In some embodiments, the transdifferentiated preadipocytes have increased expression of a pancreatic cell specific gene as compared to an unmodified preadipocyte. The pancreatic progenitor cell specific gene can be any one of RBPJ, Sox9, NeuroD, ISL1, RGS2, IL8, KCTD12, AKAP7, PCSK1, LOXL4, CPE, OAS1, PLTP, RNF128, BEX1, CEBPD, HIST2H2BE, HDAC9, KCNJ2, and combinations thereof. In some embodiments, the transdifferentiated preadipocytes can have reduced RNA expression of CD36 that is a marker for adipocyte stem cells as compared to an unmodified preadipocyte. In further embodiments, the transdifferentiated preadipocyte can have reduced RNA expression of FABP4 that is highly expressed in adipocytes manner as compared to an unmodified preadipocyte.

The transdifferentiated preadipocytes can have increased expression of a hematopoietic cell specific gene as compared to an unmodified preadipocyte cell. In some embodiments, the hematopoietic cell specific gene can be any one of CD71⁺, CHST2, AMPD3, CD58, STEAP3, MINPP1, IDH2, CD70, HLA-DRA, NX1, CD83, CD74, IFI44L, BST2, ISG15, CLIC6, PBX3, TOX2, HOPS, RAC2, RGS1, STAT4, HLA-DMB, NT5E, BTG1, CD14⁺, F13A1, FGL2, FOLR3, MX1, PSENEN, RGS1, and combinations thereof.

The transdifferentiated preadipocytes can have increased expression of a neural cell specific gene as compared to an unmodified preadipocyte. In some embodiments, the hematopoietic cell specific gene can be any one of IFI6, HS3ST2, TAGLN3, SV2B, NCALD, ENC1, COBL, EEF1A2, KBTBD11, RGL1, CA11, NETO2, DOK5, TUBB2B, PCDH19, PTPRO, SATB2, FGF13, CXADR, LPPR4, MLLT11, TMEFF2, BRSK1, NRGN, TAC1, PCDH19, CDH10, TPD52L1, HIST2H2BE, SLC16A9, MEGF10, FMNL2, EEF1A2, KIAA1598, DNER, CALB2, KAL1, and combinations thereof.

In some embodiments, the transdifferentiated cells can be provided in a cell culture vessel including flasks, tubes, and multi-well plates. The transdifferentiated cells can be provided in a cell culture vessel at any stage of transdifferentiation. In some embodiments, the cells can be seeded on a feeder layer in a cell culture vessel or on another substrate such as an extracellular matrix (synthetic or natural) composition. Other suitable substrates will be appreciated by those of skill in the art. In some embodiments, the cell culture vessel contains one or more trans-wells.

Uses of the Transdifferentiated Cells

The transdifferentiated preadipocytes described herein can be used to test compounds for their effect on a characteristic of the transdifferentiated preadipocytes. In some embodiments, the test compounds are candidate pharmaceutical compounds. In short, the transdifferentiated preadipocytes can be used in a drug or compound screening assay. As described herein are methods of evaluating a candidate compound including the step of contacting a population of transdifferentiated cells, such as pancreatic alpha cells, with an amount of a candidate compound. In some embodiments, cell growth, pancreatic cell development and differentiation, glucagon production, apoptosis, cytotoxicity, pancreatic cell replacement therapy are measured after contacting a population of transdifferentiated preadipocytes with a test compound.

EXAMPLES

Now having described the embodiments of the present disclosure, in general, the following Examples describe some additional embodiments of the present disclosure. While embodiments of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit embodiments of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.

Example 1

Introduction

Overexpression of defined transcription factors can convert one cell type into another and has important implications for regenerative medicine. Ectopic expression of key transcription factors in somatic donor cells has been used to generate many different cell types, including cells resembling blood cells [1, 2], brown fat cells [3], hepatocytes [4], sertoli cells [5], and various cell types of the neural lineage [6-9].

Expression of Oct4, partially in conjunction with Klf4, is involved in maintaining pluripotency at early developmental stages including in the blastomere, inner cell mass and epiblast. Oct4 and Klf4 have been reported as components of four defined transcription factors (Oct4, Sox2, Klf4, cMyc) that trigger reprogramming of somatic cells into pluripotent stem cells (iPS cell) that are functionally identical to embryonic stem cells (ESCs) [10]. Ectopic expression of Oct4 alone, or ectopic expression of Oct4 and Klf4, in combination with small molecule treatment, can be sufficient to generate iPS cells from neural stem cells, mouse embryonic fibroblasts (MEF) and dermal papilla cells [11-14]. Oct4 activates early epithelial-to-mesenchymal transition (EMT), which is involved in somatic cell reprogramming [15]. Szabo and colleagues observed direct conversion of human dermal fibroblasts into multipotent blood progenitors by ectopic expression of Oct4 [16]. This suggests that Oct4 can function as a haematopoietic lineage differentiation marker as well. This Example demonstrates that Oct4 and Klf4 can induce transdifferentiation of human preadipocytes into three different lineages including hematopoietic, neuronal and pancreatic endocrine cells. Furthermore, ectopic expression of Oct4 alone can induce transdifferentiation of preadipocytes into glucagon expressing pancreatic α cells.

Results

Ectopic Expression of Oct4 and Klf4 can Induce Transdifferentiation of Human Preadipocytes. Primary cultures of preadipocytes from stromal vascular fraction (SVF) of adipose tissues are a rich source of CD34 and alkaline phosphatase positive adult mesenchymal stem cells [17]. Preadipocytes can differentiate toward mesodermal lineages including osteocyte, adipocyte and chondrocyte (FIGS. 16A-16C). Two factor (OK) and 4 factor (OKSM) transfected cells were generated using highly proliferative, low passage preadipocytes (<3 passages) that had been demonstrated to have approximately 50% transfection yields using a lentiviral vector (FIGS. 17A-17D and 18). FIGS. 1 and 2A-2D show the general strategy. One day before lentiviral infection, cells were seeded on 6 well plates at a concentration of 10⁵ cells per well with Dulbecco's Modified Eagle's medium (DMEM) containing 10% FBS. For the lentiviral infection, total 25 multiplicity of infection (MOI) of lentivirus which expresses Oct4, Klf4 under control of CMV promoter (SBI and Cellomics) or polycistronic lentivirus STEMCCA which express four iPS factors (Oct4, Sox2, Klf4, cMyc) were added to the medium with 5 μg/ml polybrene (Sigma-Aldrich) (FIGS. 1 and 2A-2D).

The lentiviral infection was repeated the next day, and the culture medium was changed with fresh DMEM/5% FBS medium every other day. When cells became confluent, they were trypsinized, counted and seeded on MEF feeder layer at a density of 5×10⁴ cells/ml with mTeSR1 medium (StemCell Technology). Emerging of round cells with colony formation was observed as early as 5 days post feeder layer cultures. The two factor (OK) transfected cells showed similar morphological changes when compared to four iPS factor (OSKM) transfected cells that were undergoing early stages of somatic cell reprogramming (FIGS. 1 and 2A-2D) with the exception that more dense colony formation and immortalization from the OSKM overexpressed iPS colonies (AdiPS cells) was observed. Similar morphological changes have been shown from multi-lineage blood progenitors through overexpression of Oct4 in human adult dermal and neonatal foreskin fibroblasts [16]. Nine OK cell lines and multiple iPS cell lines were established from OSKM overexpressed preadipocytes by colony picking.

To further characterize OK transfected cells, global gene expression patterns were examined using microarray analysis (Illumina). Sample relations based on the expression of 20,909 genes showed that OK cells (single clone, mixed colonies) are distinct from preadipocyte and four factor (OSKM) induced iPS cells (AdiPS) (FIG. 3). A majority of the pluripotency genes that were highly expressed in the fully reprogrammed AdiPS cells were expressed at lower levels in two factor induced OK cells, but at levels similar to or less than levels observed in preadipocytes (FIG. 4). Expression of fibroblast and preadipocyte marker genes were expressed at lower levels in OK cells with the exception EMT activators (Slug, N-Cad) which maintained higher expression levels (FIG. 5). In contrast to a previous report [15], this suggested that OK cells underwent an epithelial-to-mesenchymal transition (EMT) rather than reprogramming processes. Epigenetic modifiers including inhibitors for histone deacetylase (HDAC), histone methyltransferase (HMT) and DNA methyltransferase (DNMT) are known to facilitate the reprogramming process by targeting epigenetic barriers [18-20]. Small molecule combination treatments (e.g., BIX-01294, BayK8644, RG-108) have been reported to enable two factor (Oct4 and Klf4) induced reprogramming of mouse embryonic fibroblasts [18]. In this Example, exposure to the DNMT1 inhibitor Zebularine did not affect reprogramming or global gene expression patterns (data not shown).

Transdifferentiation of Hematopoietic and Neuronal Lineages by Oct4 and Klf4 Overexpression. Using a BioGPS gene annotation portal (www.biogps.org), we analyzed lineage and tissue specific expression profiles of the top 292 genes characterized by a minimum 4 fold-change induced by Oct4 and Klf4 overexpression. As summarized in FIG. 6, enrichments of specific genes from three germ layers (mesoderm, ectoderm and endoderm) and inner cell mass (ICM) were observed. Overall, 41% of selected genes are annotated as tissue specific expression while the remaining is ubiquitously expressed. Specifically, a total of 45 genes are designated hematopoietic cell specific, 59 genes are brain and peripheral nerve specific, and 27 genes are exclusively expressed from pancreas and pancreatic islets. Oct4 can also function as a hematopoietic transcription factor that enhances the expression of panleukocyte marker CD45 in human fibroblasts [16]. Oct4-derived fibroblasts (CD45+ FibOct4) can differentiate into mature blood cells by additional cytokine treatments. Unlike the Oct4-derived fibroblasts (FibOct4, CD45+ FibOct4), OK cells were not observed to alter expression of hematopoietic transcription factors and cytokines (FIGS. 19-21). However, OK cells were observed to have upregulation of CD71+, early erythroid marker genes (CHST2, AMPD3, CD58, STEAP3, MINPP1, IDH2), lymphoblasts marker genes (CD70, HLA-DRA, MX1, CD83, CD74, IFI44L, BST2, ISG15, CLIC6, PBX3, TOX2, HCP5, RAC2, RGS1), lymphocyte marker genes (STAT4, HLA-DMB, NT5E, HCP5, RAC2, BTG1) and CD14+ monocyte/dendritic cell marker genes (F13A1, FGL2, FOLR3, MX1, PSENEN, RGS1) (FIG. 7).

Direct conversion of mouse fibroblasts into neurons by the combinatorial expression of neural lineage specific transcription factors (Ascl1, Brn2, Myt1l) has been demonstrated [6]. In addition, four reprogramming factor (Oct4, Sox2, Klf4, cMyc) induced intermediate cells have been identified as neural progenitor cells [21]. Tissue-specific gene expression pattern analysis revealed that OK cells expressed increased levels of brain specific markers (IFI6, HS3ST2, TAGLN3, SV2B, NCALD, ENC1, COBL, EEF1A2, KBTBD11, RGL1, CA11). Some genes are known to be exclusively expressed from fetal brain (NETO2, DOK5, TUBB2B, PCDH19, PTPRO, SATB2, FGF13, CXADR, LPPR4, MLLT11) or regions specific to the amygdala (TMEFF2, BRSK1, NRGN, TAC1P, CDH19), pineal glands (CDH10, TPD52L1, HIST2H2BE, NCALD, CXADR), prefrontal and cortex (SLC16A9, NRGN, MEGF10, FMNL2, EEF1A2, KIAA1598, KBTBD11, RGL1, CA11, SATB2), hypothalamus and thalamus (DNER, CALB2, FMNL2, KIAA1598), and spinal cord (KAL1, DNER, MEGF10). Most of the brain specific genes that were highly induced in OK cells were undetectable or at very low levels when compared to untransfected human preadipocytes (FIG. 8). Neuronal differentiation, however, did not proceed any further: OK cells did not show neuronal morphological changes such as neuronal rosettes or nerve fibers (axon) (data not shown). In addition, the expression of neural lineage-specific transcription factors (Ascl1, Brn2, Myt1l) were not altered in OK cells. Without being bound by theory, these results suggest that Oct4 and Klf4 induced neural transdifferentiation can be independent of neural development, and that Oct4 and Klf4 overexpression directly convert mesoderm to ectoderm lineages.

Transdifferentiation of Pancreatic α Cells by Oct4/Klf4 or Oct4 Alone. As a part of an endocrine organ, pancreatic α and β cells play vital roles maintaining blood glucose homeostasis by secreting glucagon and insulin, respectively. Three of pancreatic lineage specific transdifferentiation factors (Pdx1, Ngn3, Mafa) have been identified that efficiently convert adult hepatocytes and pancreatic exocrine cells into insulin secreting pancreatic β cells [22-25]. However, pancreatic α cell specific transdifferentiation has not been reported. Global gene expression analysis indicated that 27 pancreatic islet specific genes were enriched in OK cells including pancreatic progenitor markers (RBPJ, Sox9, NeuroD and ISL1) (FIG. 9). The enriched genes are functionally associated with G protein related cell signaling and which are responsible for glucose sensing (RGS2, 1L8, KCTD12, AKAP7), transcription regulation (ISL1, BEX1, CEBPD, HIST2H2BE, NEUROD, SOX9, RBPJ) and enzymes for pancreatic hormone processing (PCSK1, LOXL4, CPE, OAS1, PLTP, RNF128). Among the four pancreatic hormones that secret from distinct cells in the pancreatic islets, upregulation of glucagon (GCG) gene (FIG. 9) was observed, but not insulin, somatostatin, or pancreatic polypeptide (data not shown). Without being bound by theory, these results suggest that human preadipocytes can be directly converted to pancreatic α cells by Oct4 and Klf4 overexpression.

Pancreatic cell specific gene expression has been confirmed with real-time RTPCR using SYBR green conjugated gene specific primers that show distinct expression patterns across three types of pancreatic cells (α, β and exocrine cells)[26]. As shown in FIGS. 10A-10O, the expression of pancreatic α cell markers (GCG, CNTN1, PCSK1, PDK4, RGS4, IRX2, LPPR4, LOXL2, KCTD12, KL) and β cell markers (ISL1, HDAC9, KCNJ2) were been upregulated dramatically in OK cells while the preadipocyte marker gene (CD36, FABP4) expression decreased (FIGS. 10A-10O). The expression of pancreatic exocrine cell specific markers (AMY1C, AMT1B, AMY1A) were highly expressed in all samples (data not shown). In order to assess glucagon and NeuroD protein expression (a pancreatic α cell product and a pancreatic progenitor marker, respectively), OK cells were seeded onto matrigel coated cell culture plates and cells were examined by immunocytochemistry. The results demonstrate that all of the colonies on matrigel stained with specific antibodies against glucagon or NeuroD except for the feeder cells (FIGS. 11A-11D). To evaluate the secretion of glucagon from the transdifferentiated pancreatic α cells (OK cells, Oct4 alone) using an ELISA from the cell culture supernatants. Although the expression of glucagon from the cell extracts was detected, glucagon secretion was barely detectable. Without being bound by theory, this suggests that pancreatic α cells are missing a component for the glucagon processing and secretion while the cells highly expresses pancreatic exocrine cell markers such as AMY1C, AMT1B, AMY1A.

Analysis of the regulatory transcription factor binding sites indicated that the promoter region of most genes that were upregulated in OK cells contain putative POU domain binding sequences, but not Klf4 binding sequences, suggesting Klf4 might be dispensable for the transdifferentiation processes. To test this hypothesis, preadipocytes were transfected with Oct4 only using a lentiviral vector and repeated the experiment using the same conditions used previously. The general outline is shown in FIGS. 12 and 13A-13D. Similar to the previous two factor overexpression, colony formation was observed at 5 days after culturing on feeder cell layer. The cells had virtually identical morphology as the two factor (Oct4, Klf4) induced OK cells (FIGS. 12 and 13A-13D). Next, the pancreatic cell specific gene and protein expression from established colonies (Oct4#1, Oct4#5) and mixed colonies (Oct4) was analyzed. As shown in FIGS. 14A-14N, the expression of pancreatic cell markers from Oct4 single factor overexpressed cells was upregulated at levels similar to two factor (Oct4, Klf4) induced OK cells, except for LOXL2, RGS4 and PCSK1. Glucagon and NeuroD protein expression was confirmed by immunohistochemistry (FIGS. 15A-15D). Without being bound by theory, these results suggest that ectopic expression of Oct4 alone is sufficient to induce transdifferentiation of human preadipocytes into glucagon expressing pancreatic α cells. These results are consistent with previous findings that Oct4 and Nanog play roles in maintaining pluripotency while Klf4 and cMyc regulate cellular division [27].

This Example also indicates that Klf4 is dispensable in the transdifferentiation processes. The Oct4 induced pancreatic α cells was expanded for more than 10 passages without any loss of cellular proliferation properties or pancreatic marker gene expression (data not shown). Glucagon producing pancreatic α cells comprise 0.2-0.5% of pancreatic islets and show great plasticity to become insulin secreting β cells after severe β cell loss in mouse model[28]. Furthermore, Bramswig and colleagues demonstrated that histone methyltransferase inhibitor treatment facilitates the conversion of pancreatic α cell to insulin secreting β cells[26]. These findings regarding the plasticity of adipose stem cells and transdifferentiation into pancreatic endocrine cells and the development of a simple and efficient method to produce pancreatic α cells will provide a model to advance therapeutic strategies for metabolic disease.

Methods

Cell Culture and Lentiviral Transfection.

Human preadipocytes (LaCell, LA) were maintained with Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS, 50 U/ml penicillin and 50 μg/ml streptomycin at 37° C., 5% CO2 in a humidified incubator. High titer lentivirus, overexpressing human Oct4, Klf4 or polycistronic lentivirus which express four iPS factors (STEMCCA), was purchased from System Biosciences (San Diego, Calif.), Cellomics (Halethorpe, Md.) and EMD Millipore (Temecula, Calif.). The day before lentiviral transfection, human preadipocytes were trypsinized, counted, and seeded in 6-well plates at a density of 10⁵ cells/well. The next day, culture medium was replaced with pre-warmed medium containing 5 μg/ml polybrene (Sigma-Aldrich) and 25 MOI of lentivirus. Lentiviral infection was repeated the next day, and culture medium was changed with fresh preadipocyte growth medium. When the cells reached confluence, cells were trypsinized, counted and seeded on a prepared MEF feeder layer at a density of 5×10⁴ cells with mTeSR1 medium (StemCell Technology).

Microarray and Gene Expression Analysis. Total RNA was prepared from cultures using Trizol Reagent (Life Technology) and the RNeasy Mini RNA isolation kit (Qiagen, Valencia, Calif.) with DNase I digestion. The RNA quality was verified using an Agilent Bioanalyzer 2100 (Agilent Technologies; Palo Alto, Calif.) using the RNA 6000 Pico Assay. Generation of double-stranded cDNA, preparation and labeling of cRNA, hybridization to HumanHT-12 v4 Expression BeadChip (Illumina, San Diego, Calif.), washing, and scanning were all performed according to the standard Illumina protocol. Quantitative PCR to measure mRNA expression levels was performed with PrimeTime qPCR assays (Integrated DNA Technologies, Coralville, Iowa) using a 7700 real-time PCR system in the genomic core facility at Pennington Biomedical Research Center. Expression levels were compared to known standard samples and were normalized GAPDH.

Immunocytochemistry. Cells were fixed with 4% paraformaldehyde in PBS for 10 min, and incubated for 1 hr with antibodies specific for glucagon and NeuroD1 (Abcam). After washing three times with PBS, cells were incubated for 1 hr with fluorescent conjugated secondary antibody (Invitrogen). Nuclei were detected by DAPI staining (Vector shield).

Abbreviations Used in the Examples

-   AdiPS=Adipose derived iPS cell -   DNMT=DNA methyltransferase -   EMT=epithelial-to-mesenchymal transition -   ESCs=embryonic stem cells -   HDAC=histone deacetylase -   HMT=histone methyltransferase -   ICM=inner cell mass -   iPS cell=induced pluripotent stem cell -   MEF=mouse embryonic fibroblasts -   OK=Oct4, Klf4 -   OKSM=Oct4, Klf4, Sox2, cMyc -   SVF=stromal vascular fraction

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TABLE 1 SEQ ID GenBank NO Accession Number Gene Name  1 NM_203289 OCT4  2 NM_001173531 OCT4  3 NM_001285986 OCT4  4 NM_001285987.1 OCT4  5 NM_002701 OCT4  6 NM_001252452 OCT4  7 NM_013633 OCT4  8 NP_001272916.1 OCT4  9 NP_002692.2 OCT4  10 NP_001272915.1 OCT4  11 NP_038661.2 OCT4  12 NM_001314052 Klf4  13 NM_010637 Klf4  14 NP_004226.3 Klf4  15 NP_001300981.1 Klf4  16 NP_034767.2 Klf4  17 NM_003106 Sox2  18 NM_011443 Sox2  19 NP_003097.1 Sox2  20 NP_035573.3 Sox2  21 NM_002467 cMyc  22 NM_001177352 cMyc  23 NM_001177353 cMyc  24 NM_001177354 cMyc  25 NM_010849 cMyc  26 NP_002458.2 cMyc  27 NP_001170823.1 cMyc  28 NP_001170824.1 cMyc  29 NP_001170825.1 cMyc  30 NM_001256063 CNTN1  31 NM_001256064 CNTN1  32 NM_001843 CNTN1  33 NM_175038 CNTN1  34 NM_001159647 CNTN1  35 NM_001159648 CNTN1  36 NP_001242992.1 CNTN1  37 NP_001242993.1 CNTN1  38 NP_001834.2 CNTN1  39 NP_778203.1 CNTN1  40 NP_001153119.1 CNTN1  41 NP_001153120.1 CNTN1  42 NP_031753.1 CNTN1  43 NM_001177876.1 PCSK1  44 NM_000439 PCSK1  45 NM_001177875 PCSK1  46 NM_013628 PCSK1  47 NP_000430.3 PCSK1  48 NP_001171346.1 PCSK1  49 NP_038656.1 PCSK1  50 NM_002612 PDK4  51 NP_002603.1 PDK4  52 NM_001102445 RGS4  53 NM_001113380 RGS4  54 NM_001113381 RGS4  55 NM_005613 RGS4  56 NM_009062 RGS4  57 NP_001095915.1 RGS4  58 NP_001106851.1 RGS4  59 NP_001106582.1 RGS4  60 NP_005604.1 RGS4  61 NP_033088.2 RGS4  62 NM_001134222.1 IRX2  63 NM_033267.4 IRX2  64 NM_010574 IRX2  65 NP_001127694.1 IRX2  66 NP_150366.1 IRX2  67 NP_034704.1 IRX2  68 NM_001166252.1 LPPR4  69 NM_014839.4 LPPR4  70 NP_001159724.1 LPPR4  71 NP_055654.2 LPPR4  72 NM_002318.2 LOXL2  73 NP_002309.1 LOXL2  74 NM_032211.6 LOXL4  75 NP_115587.6 LOXL4  76 NM_138444.3 KCTD12  77 NP_612453.1 KCTD12  78 NM_004795.3 KL  79 NP_004786.2 KL  80 NM_005349.3 RBPJ  81 NM_015874.4 RBPJ  82 NM_203283.2 RBPJ  83 NM_203284.2 RBPJ  84 NP_005340.2 RBPJ  85 NP_056958.3 RBPJ  86 NP_976028.1 RBPJ  87 NP_976029.1 RBPJ  88 NM_000346.3 Sox9  89 NP_000337.1 Sox9  90 NM_002500.4 NeuroD  91 NP_002491.2 NeuroD  92 NM_002202.2 ISL1  93 NP_002193.2 ISL1  94 NM_002923.3 RGS2  95 NP_002914.1 RGS2  96 NM_000584.3 IL8  97 NP_000575.1 IL8  98 NM_004842.3 AKAP7  99 NM_016377.3 AKAP7 100 NM_138633.2 AKAP7 101 NP_004833.1 AKAP7 102 NP_057461.2 AKAP7 103 NP_619539.1 AKAP7 104 NM_001873.3 CPE 105 NP_001864.1 CPE 106 NM_001032409.2 OAS1 107 NM_001320151.1 OAS1 108 NM_002534.3 OAS1 109 NM_016816.3 OAS1 110 NP_001027581.1 OAS1 111 NP_001307080.1 OAS1 112 NP_002525.2 OAS1 113 NP_058132.2 OAS1 114 NM_001242920.1 PLTP 115 NM_001242921.1 PLTP 116 NM_006227.3 PLTP 117 NM_182676.2 PLTP 118 NP_001229849.1 PLTP 119 NP_001229850.1 PLTP 120 NP_006218.1 PLTP 121 NP_872617.1 PLTP 122 NM_001199017.2 PGS4 123 NM_001199018.2 PGS4 124 NM_001284357.1 PGS4 125 NM_017691.4 PGS4 126 NP_001185946.1 PGS4 127 NP_001185947.1 PGS4 128 NP_001271286.1 PGS4 129 NP_060161.2 PGS4 130 NM_024539.3 RNF128 131 NM_194463.1 RNF128 132 NP_078815.3 RNF128 133 NP_919445.1 RNF128 134 NM_018476.3 BEX1 135 NP_060946.3 BEX1 136 NM_001204144.2 HDAC9 137 NM_001204145.2 HDAC9 138 NM_001204146.2 HDAC9 139 NM_001204147.2 HDAC9 140 NM_001204148.2 HDAC9 141 NP_001191073.1 HDAC9 142 NP_001191074.1 HDAC9 143 NP_001191075.1 HDAC9 144 NP_001191076.1 HDAC9 145 NP_001191077.1 HDAC9 146 NP_001308797.1 HDAC9 147 NP_001308798.1 HDAC9 148 NP_001308799.1 HDAC9 149 NP_001308800.1 HDAC9 150 NP_001308801.1 HDAC9 151 NP_001308802.1 HDAC9 152 NP_001308803.1 HDAC9 153 NP_001308804.1 HDAC9 154 NP_001308805.1 HDAC9 155 NP_001308806.1 HDAC9 156 NP_001308807.1 HDAC9 157 NP_001308808.1 HDAC9 158 NP_001308813.1 HDAC9 159 NP_001308814.1 HDAC9 160 NP_001308815.1 HDAC9 161 NP_001308816.1 HDAC9 162 NP_001308817.1 HDAC9 163 NP_001308818.1 HDAC9 164 NP_001308819.1 HDAC9 165 NP_001308820.1 HDAC9 166 NP_001308822.1 HDAC9 167 NP_001308823.1 HDAC9 168 NP_001308824.1 HDAC9 169 NP_001308825.1 HDAC9 170 NP_001308826.1 HDAC9 171 NP_001308827.1 HDAC9 172 NP_001308828.1 HDAC9 173 NP_001308829.1 HDAC9 174 NP_001308830.1 HDAC9 175 NP_001308831.1 HDAC9 176 NP_055522.1 HDAC9 177 NP_478056.1 HDAC9 178 NP_848510.1 HDAC9 179 NP_848512.1 HDAC9 180 NM_000891.2 KCNJ2 181 NP_000882.1 KCNJ2 182 NM_000072.3 CD36 183 NM_001001547.2 CD36 184 NM_001001548.2 CD36 185 NM_001127443.1 CD36 186 NM_001127444.1 CD36 187 NM_001289908.1 CD36 188 NM_001289909.1 CD36 189 NM_001289911.1 CD36 190 NP_000063.2 CD36 191 NP_001001547.1 CD36 192 NP_001001548.1 CD36 193 NP_001120915.1 CD36 194 NP_001120916.1 CD36 195 NP_001276837.1 CD36 196 NP_001276838.1 CD36 197 NP_001276840.1 CD36 198 NM_001442.2 FABP4 199 NP_001433.1 FABP4 200 NM_001128148.2 CD71 201 NM_001313965.1 CD71 202 NM_001313966.1 CD71 203 NM_003234.3 CD71 204 NP_001121620.1 CD71 205 NP_001300894.1 CD71 206 NP_001300895.1 CD71 207 NP_003225.2 CD71 208 NM_004267.4 CHST2 209 NP_004258.2 CHST2 210 NM_000480.2 AMPD3 211 NM_001025389.1 AMPD3 212 NM_001025390.1 AMPD3 213 NM_001172430.1 AMPD3 214 NM_001172431.1 AMPD3 215 NP_000471.1 AMPD3 216 NP_001020560.1 AMPD3 217 NP_001020561.1 AMPD3 218 NP_001165901.1 AMPD3 219 NP_001165902.1 AMPD3 220 NM_001144822.1 CD58 221 NM_001779.2 CD58 222 NP_001138294.1 CD58 223 NP_001770.1 CD58 224 NM_001178117.1 MINPP1 225 NM_001178118.1 MINPP1 226 NM_004897.4 MINPP1 227 NP_001171588.1 MINPP1 228 NP_001171589.1 MINPP1 229 NP_004888.2 MINPP1 230 NM_001289910.1 IDH2 231 NM_001290114.1 IDH2 232 NM_002168.3 IDH2 233 NP_001276839.1 IDH2 234 NP_001277043.1 IDH2 235 NP_002159.2 IDH2 236 NM_001252.4 CD70 237 NP_001243.1 CD70 238 NM_019111.4 HLA-DRA 239 NP_061984.2 HLA-DRA 240 NM_001135659.2 NX1 241 NM_001320156.2 NX1 242 NM_001320157.3 NX1 243 NM_004801.5 NX1 244 NM_138735.4 NX1 245 NP_001129131.1 NX1 246 NP_001307085.1 NX1 247 NP_001307086.1 NX1 248 NP_004792.1 NX1 249 NP_620072.1 NX1 250 NM_001040280.1 CD83 251 NM_001251901.1 CD83 252 NM_004233.3 CD83 253 NP_001035370.1 CD83 254 NP_001238830.1 CD83 255 NP_004224.1 CD83 256 NM_001025158.2 CD74 257 NM_001025159.2 CD74 258 NM_004355.3 CD74 259 NP_001020329.1 CD74 260 NP_001020330.1 CD74 261 NP_004346.1 CD74 262 NM_006820.3 IFI44L 263 NP_006811.2 IFI44L 264 NM_004335.3 BST2 265 NP_004326.1 BST2 266 NM_005101.3 ISG15 267 NP_005092.1 ISG15 268 NM_001317009.1 CLIC6 269 NM_053277.2 CLIC6 270 NP_001303938.1 CLIC6 271 NP_444507.1 CLIC6 272 NM_001134778.1 PBX3 273 NM_006195.5 PBX3 274 NP_001128250.1 PBX3 275 NP_006186.1 PBX3 276 NM_001098796.1 TOX2 277 NM_001098797.1 TOX2 278 NM_001098798.1 TOX2 279 NM_032883.2 TOX2 280 NP_001092266.1 TOX2 281 NP_001092267.1 TOX2 282 NP_001092268.1 TOX2 283 NP_116272.1 TOX2 284 NR_040662.1 HCP5 285 NM_002872.4 RAC2 286 NP_002863.1 RAC2 287 NM_002922.3 RGS1 288 NP_002913.3 RGS1 289 NM_001243835.1 STAT4 290 NM_003151.3 STAT4 291 NP_001230764.1 STAT4 292 NP_003142.1 STAT4 293 NM_002118.4 HLA-DMB 294 NP_002109.2 HLA-DMB 295 NM_001204813.1 NT5E 296 NM_002526.3 NT5E 297 NP_001191742.1 NT5E 298 NP_002517.1 NT5E 299 NM_001731.2 BTG1 300 NP_001722.1 BTG1 301 NM_000591.3 CD14 302 NP_000582.1 CD14 303 NM_000129.3 F13A1 304 NP_000120.2 F13A1 305 NM_006682.2 FGL2 306 NP_006673.1 FGL2 307 NM_000804.3 FOLR3 308 NP_000795.2 FOLR3 309 NM_001144925.2 MX1 310 NP_001138397.1 MX1 311 NM_172341.2 PSENEN 312 NP_758844.1 PSENEN 313 NM_002038.3 IFI6 314 NP_002029.3 IFI6 315 NM_006043.1 HS3ST2 316 NP_006034.1 HS3ST2 317 NM_013259.2 TAGLN3 318 NP_037391.2 TAGLN3 319 NM_014848.6 SV2B 320 NP_055663.1 SV2B 321 NM_001040624.1 NCALD 322 NP_001035714.1 NCALD 322 NP_001035714.1 NCALD 323 NM_003633.3 ENC1 324 NP_003624.1 ENC1 325 NM_001287436.1 COBL 326 NP_001274365.1 COBL 327 NM_001958.3 EEF1A2 328 NP_001949.1 EEF1A2 329 NM_001958.3 KBTBD11 330 NP_055682.1 KBTBD11 331 NM_015149.4 RGL1 332 NP_055964.3 RGL1 333 NM_001217.4 CA11 334 NP_001208.2 CA11 335 NM_018092.4 NETO2 336 NP_060562.3 NETO2 337 NM_018431.4 DOK5 338 NP_060901.2 DOK5 339 NM_178012.4 TUBB2B 340 NP_821080.1 TUBB2B 341 NM_001105243.1 PCDH19 342 NP_001098713.1 PCDH19 343 NM_030667.2 PTPRO 344 NP_109592.1 PTPRO 345 NM_001172509.1 SATB2 346 NP_001165980.1 SATB2 347 NM_004114.3 FGF13 348 NP_004105.1 FGF13 349 NM_001338.4 CXADR 350 NP_001329.1 CXADR 351 NM_006818.3 MLLT11 352 NP_006809.1 MLLT11 353 NM_016192.3 TMEFF2 354 NP_057276.2 TMEFF2 355 NM_032430.1 BRSK1 356 NP_115806.1 BRSK1 357 NM_006176.2 NRGN 358 NP_006167.1 NRGN 359 NM_003182.2 TAC1 360 NP_003173.1 TAC1 361 NM_006727.4 CDH10 362 NP_006718.2 CDH10 363 NM_003287.3 TPD52L1 364 NP_003278.1 TPD52L1 365 NM_003528.2 HIST2H2BE 366 NP_003519.1 HIST2H2BE 367 NM_194298.2 SLC16A9 368 NP_919274.1 SLC16A9 369 NM_032446.2 MEGF10 370 NP_115822.1 MEGF10 371 NM_052905.3 FMNL2 372 NP_443137.2 FMNL2 373 NM_001127211.2 KIAA1598 374 NP_001120683.1 KIAA1598 375 NM_139072.3 DNER 376 NP_620711.3 DNER 377 NM_000216.3 KAL1 378 NP_000207.2 KAL1 379 NM_002838.4 CD45 380 NP_002829.3 CD45 381 NM_001008219.1 AMY1C 382 NP_001008220.1 AMY1C 383 NM_004038.3 AMY1A 384 NP_004029.2 AMY1A 385 NM_002054.4 GCG 386 NP_002045.1 GCG 387 NM_005195.3 CEBPD 388 NP_005186.2 CEBPD 389 NM_182915.2 STEAP3 390 NP_878919.2 STEAP3 391 XM_001692619.1 AMT1B 392 XM_001692619.1 AMT1B 

We claim:
 1. A method of transdifferentiating mammalian preadipocytes, the method comprising: (a) culturing mammalian preadipocytes; (b) infecting the mammalian preadipocytes with a viral vector encoding Oct4 such that the mammalian preadipocytes overexpress Oct4; (c) culturing the infected cells until about 100% confluent; (d) subculturing the cells of step (c), wherein subculturing comprises seeding at least a portion of the cells onto a mouse embryonic fibroblast (MEF) feeder cell layer; (e) expanding the cells seeded onto the feeder layer of step (d) for a period of time sufficient to produce a population of cells which produce glucagon and which express a pancreatic alpha cell marker gene selected from the group consisting of CGC, CNTN1, PCSK1, PDK4, RGS4, IRX2, LPPR4, LOXL2, KCTD12, KL, and any combination thereof.
 2. The method of claim 1, further comprising overexpressing exogneous Klf4 in at least one of the mammalian preadipocytes.
 3. The method of claim 2, wherein the exogenous Klf4 is simultaneously overexpressed with exogenous Oct4 in the same mammalian preadipocyte.
 4. The method of claim 2, wherein overexpression of exogenous Klf4 occurs via transfection or transduction of the at least one mammalian preadipocyte with an exogenous Klf4.
 5. The method of claim 1, further comprising overexpressing exogenous Sox2 in at least one of the mammalian preadipocytes.
 6. The method of claim 5, wherein overexpression of exogenous Sox2 occurs via transfection or transduction of the at least one mammalian preadipocyte with an exogenous Sox2.
 7. The method of claim 1, further comprising overexpressing exogenous cMyc at least one of the mammalian preadipocytes.
 8. The method of claim 7, wherein exogenous cMyc is simultaneously overexpressed with exogenous Oct4 in the same mammalian preadipocyte.
 9. The method of claim 1, wherein the mammalian preadipocytes have been passaged 3 times or less prior to overexpression of exogenous Oct4.
 10. The method of claim 1, wherein the cells seeded onto the feeder layer are expanded on the feeder layer for about 3, 4, 5, 6, 7, 8, 9, or about 10 days.
 11. The method of claim 1, wherein culturing the population of cells is performed without passaging for about 1,2,3, 4, or 5 days prior to performing subculturing of cells.
 12. The method of claim 1, further comprising the step of contacting the population of cells of producing glucagon with a compound.
 13. The method of claim 12, further comprising the step of evaluating a characteristic of the cells producing glucagon, wherein the characteristic is selected from the group consisting of cell growth, cell development, cell differentiation, glucagon production, apoptosis, cytotoxicity, and any combination thereof.
 14. The method of claim 1, further comprising overexpressing exogenous Kif 4, exogenous cMyc, and exogenous Sox2 in the at least one mammalian preadipocyte.
 15. The method of claim 1, wherein expanding the cells is performed using a serum free media.
 16. The method of claim 1, wherein the cells producing glucagon express NeuroD. 